This invention relates to a novel cytoplasmic post-prolyl dipeptidase, and its role in regulating apoptosis, or programmed cell death (PCD).
Apoptosis is a physiological form of cell death, and it shapes a number of diverse biological processes, such as development and homeostasis. It occurs in response to diverse stimuli which fall into two categories. The first is activation induced cell death following specific stimulation; and the second is death by neglect after withdrawal of a life-promoting stimulation, such as a growth factor, serum or cell-cell contact. While these two types of PCD take place under very different circumstances, both depend on the activation of caspases, a family of cysteine proteases which are present in the cytoplasm of cells as inactive proenzymes.
Apoptotic stimuli lead to the activation of certain caspases by specific proteolytic cleavage, enabling them to activate other caspases through a proteolytic cascade, which eventually leads to cell death. Studies of activation-induced cell death through the Fas/TNF receptors have implicated the death effector domain containing protease FLICE (caspase 8) in the initiation of the caspase cascade. However, while most cells contain all the components of the apoptotic machinery, and are susceptible to PCD by neglect, no caspase-cascade regulator induced under these conditions, has been identified.
With respect to apoptosis in the nervous system, recent studies have shown that neuronal cell death resulting from degeneration or trauma occurs by the process of apoptosis, the biochemical hallmarks of which include cytolemmal membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering. Thus, given the role of apoptosis in neural degeneration, it would be highly desirable to have a process for identifying compounds that modulate this apoptosis. Such compounds could be effective as therapeutic agents in the treatment or prevention of neural conditions associated with inappropriate neural degeneration. However, one of the fundamental technological problems associated with the identification of these compounds is the lack of suitable targets to use in screening for regulators of apoptosis. Thus, there is a need to identify new enzymatic targets as a first step in developing new therapeutic compounds.
We have discovered a novel, cytoplasmic post-prolyl dipeptidase, which has similarities to, but is distinct from, the membrane-bound T-cell serine protease CD26. This enzyme is expressed in quiescent, non-cycling T-cells and in neurons, and is called quiescent cell post-prolyl dipeptidase (QPP; previously termed DPIVb).
In a first aspect, the invention features a substantially pure, recombinant quiescent cell post-prolyl dipeptidase polypeptide (QPP), wherein the polypeptide inhibits quiescent cells from undergoing apoptosis. Preferably, the polypeptide comprises the amino acid sequence of SEQ ID NO 6.
In a second and related aspect, the invention features a substantially pure nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid sequence comprises SEQ ID NO 5, or a sequence that is complementary to a sequence that hybridizes to SEQ ID NO 5 under stringent hybridization conditions encoding a QPP polypeptide, or encodes a polypeptide of SEQ ID NO 6.
The invention also features a host cell, for example, yeast, E. coli, Chinese hamster ovary (CHO), or fibroblast, transformed or transfected with a transgene that includes a nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid molecule is operably linked to a regulatory sequence, and wherein the nucleic acid sequence comprises SEQ ID NO 5, or a sequence that is complementary to a sequence that hybridizes to SEQ ID NO 5 under stringent hybridization conditions, or encodes a polypeptide of SEQ ID NO 6.
In a related aspect, the invention features a method of producing a QPP polypeptide including transforming or transfecting a cell with a nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid molecule is operably linked to a regulatory sequence, and further wherein the nucleic acid sequence comprises SEQ ID NO 5, or a sequence that is complementary to a sequence that hybridizes to SEQ ID NO 5 under stringent hybridization conditions, or encodes a polypeptide of SEQ ID NO 6; culturing the cell under conditions for expressing the nucleic acid; and isolating the QPP polypeptide.
The purified QPP sequences of the invention have a number of utilities. The invention features methods for: 1) identifying a therapeutic candidate compound that modulates a QPP effect in quiescent cells, which includes contacting a recombinant QPP polypeptide, preferably, the QPP polypeptide of SEQ ID NO 6, or a transformed or transfected cell encoding and expressing a recombinant QPP polypeptide, with the compound and measuring QPP biological activity; 2) inhibiting apoptosis in a quiescent cell, which includes contacting the cell with a recombinant QPP polypeptide, preferably, the QPP polypeptide of SEQ ID NO 6, in an amount sufficient to protect the cell from death when an apoptosis stimulus is administered; and 3) treating a mammal for a condition related to quiescent cell death, that includes administering to the mammal a recombinant QPP polypeptide, preferably, the QPP polypeptide of SEQ ID NO 6, in an amount sufficient to inhibit the cell death. Preferably, the condition is a neurodegenerative disorder or an immune system disorder, and the QPP polypeptide may be administered by transplanting a host cell into the mammal, wherein the host cell is ex vivo transfected with a transgene comprising a nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid molecule is operably linked to a regulatory sequence and is expressed in the transplanted host cell.
The recombinant QPP sequences can also be used to make antibodies (polyclonal, monoclonal, or recombinant) using conventional methods, involving immunization of, e.g., rabbits, mice, or human volunteers. The antibodies can be used in standard ELISA assays to measure QPP levels in patients being tested for diseases which potentially involve increased or decreased QPP levels; for example, HIV patients, who have lost QPP-containing T-cells, will exhibit decreased QPP levels, with the QPP concentration being diagnostic of the stage of the disease. Generally, because QPP is a cytoplasmic enzyme, the assay is carried out on peripheral blood lymphocyte samples which have first been treated to lyse T-cells to release the enzyme.
By xe2x80x9csubstantially purexe2x80x9d is meant a polypeptide or nucleic acid that has been separated from components which normally accompany it, or which accompany it upon recumbent expression. An enzyme is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably, at least 90%, and most preferably, at least 99%, pure QPP by weight. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By xe2x80x9capoptosisxe2x80x9d is meant the process of programmed cell death wherein a dying cell displays a set of well-characterized biochemical hallmarks which include cytolemmal membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering.
By xe2x80x9cstringent hybridization conditionsxe2x80x9d is meant incubation conditions that destabilize mismatched heteroduplexes. Conditions for high stringency hybridization, such as for PCR, Northern, Southern, in situ hybridization, or DNA sequencing, are described, for example, in F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1994.
By xe2x80x9ctransformedxe2x80x9d or xe2x80x9ctransfectedxe2x80x9d cell is meant a cell in which foreign nucleic acid molecules have been introduced. Lipofection, DEAE-dextranmediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the methods known to those skilled in the art which may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., and mitochondria and chloroplasts), bacteria, yeast, fungi, algae, animal tissue, and cultured cells.
By xe2x80x9capoptotic stimulusxe2x80x9d is meant any chemical or physical treatment which initiates apoptosis as defined above. For example, nerve growth factor withdrawal, hypoxia, exposure to staurosporine, and cerebral ischemia are stimuli capable of inducing apoptosis in neurons.
The xe2x80x9cquiescent cellxe2x80x9d is meant a non-cycling cell in a non-replicative or resting state.
By xe2x80x9cQPP effectxe2x80x9d is meant QPP""s VbP-sensitive protection against apoptosis in quiescent cells.
By xe2x80x9ctest compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate cell death, by employing one of the assay methods described herein. Test compounds may include, for example, small molecules, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By xe2x80x9ctherapeutic candidate compoundxe2x80x9d is meant a compound that modulates a QPP effect, and is, therefore, a candidate for use as a therapeutic to inhibit cell death in quiescent cells.
By xe2x80x9cmeasuring QPP biological activityxe2x80x9d is meant analyzing the effect of a test compound, whether the effects be chemical or physical. The material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be for the purpose of detecting a change in QPP biology such as altered RNA stability, altered protein stability, altered protein levels, altered enzymatic activity, or an altered rate of QPP-mediated apoptotic death. The means for analyzing may include, for example, antibody labeling, immunoprecipitation, substrate cleavage assays, and methods known to those skilled in the art for detecting nucleic acids.
By xe2x80x9cmodulatesxe2x80x9d is meant changing, either by decrease or increase.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence are connected in such a way as to permit expression of the gene product under the control of the regulatory sequence.
By a xe2x80x9ctransgenexe2x80x9d is meant a nucleic acid sequence which is inserted by artifice into a cell and becomes a part of the genome of that cell and its progeny. Such a transgene may be partly or entirely heterologous to the cell.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.